Alzheimer's disease (AD) is a worldwide, incurable disease, and the most common form of dementia. Numbers of cases are rising, and since its discovery the only approved medications have treated only the symptoms, not the pathological cause. With the cost to society rising, the debilitating nature of the disease and the pressure put on the family members and support network of patients, disease modifying therapies are in dire need. Current models have proven an invaluable tool with which to study certain aspects of the disease and the genetics behind it, however the lack of clinically approved medications in the last 20 years suggests new models are needed. Based on the amyloid cascade hypothesis, this thesis initially characterises two models of β-Amyloid oligomer (Aβo) induced cognitive deficits. Both models are created by ICV injection of soluble Aβo into the brain of rat. The models differ only by the molecular weight of the Aβo 1-42, one, referred to as low molecular weight (LMW) Aβo, with stable dimers, trimers and tetramers, the other, referred to as high molecular weight (HMW) Aβo, consisting of assemblies ranging from ~50 to ~150 kDa. It was found that behavioural deficits were similar between the two, with a robust object recognition deficit, but no working memory deficit. Both models also showed a deficit in the synaptic marker PSD-95; however the LMW Aβo caused a deficit in the frontal cortex, whereas the HMW Aβo caused a hippocampal deficit. The role of the cellular prion protein (PrPC) was explored, by blocking its binding to Aβo with the antibody 6D11. Interestingly the two models showed different results. The HMW Aβo deficits were completely blocked by the 6D11 application, however the LMW Aβo deficits were only partially prevented. Finally, Fasudil, a vasodilator approved in parts of Asia, was used to inhibit Rho-kinase, showing a prevention of the cognitive deficits in the HMW Aβo model. The results of this thesis show the ICV administration of Aβo to be a useful model for investigating the effects of Aβo, provides a platform with which to study the differing effects of Aβo with different oligomeric assemblies, and a model to test therapeutic strategies with relevance to AD.

Bones of the cranial vault protect the underlying brain but preserve flexibility to allow brain growth. The edges of skull bones are joined by fibrous sutures, which provide structural support and are the sites of bone growth. Craniosynostosis (CS), premature fusion of bones at the sutures, causes skull deformity and secondary problems in brain development. Bone morphogenetic protein (BMP) signaling is critical in regulating osteoblast differentiation and ossification. Several components of the BMP signaling pathway are associated with increased risk of CS, supporting a central role for BMP signaling in suture formation and development of CS. However, the specific factors involved and disease mechanisms remain largely unknown. To address the gaps in our knowledge, we identified regulatory elements active in skeletal tissues associated with the risk of CS. Briefly, we selected conserved noncoding elements within the genomic regions near the CS risk genes, BMP2 and BMPER (BMP Binding Endothelial Regulator), and performed zebrafish transgenesis to screen for enhancer activities in cranial skeletal tissues. We found multiple enhancers that directed transgene expression consistent with the expression of endogenous bmp2 and bmper genes. Using confocal microscopy, we demonstrated activity of the enhancer, -707BMPER, in cartilage closely associated with developing frontal bones, suggesting its involvement in cranial bone growth, suture formation and the risk of CS. We also performed an enhanced yeast one-hybrid (eY1H) assay to determine the transcription factors that interacted with the identified enhancers, implicating the underlying signaling pathways in regulation of their activity. Compelling human genetic evidence has revealed a role for SMAD6 (mothers against decapentaplegic homolog 6), a negative regulator of BMP signaling, in craniosynostosis. SMAD6 mutations are also associated with cardiovascular abnormalities and nonsyndromic radioulnar synostosis. However, there are significant unanswered questions about the mechanisms linking specific SMAD6 mutations to any of these defects. To create a tractable animal model to address these questions, we used CRISPR targeting to mutate the zebrafish ortholog, smad6a and smad6b. In addition, we developed a zebrafish assay to evaluate the functional consequences of SMAD6 sequence variants, based on the ability of Smad6 to disrupt dorsal-ventral patterning of zebrafish embryos in a dose-dependent manner. We anticipate that the zebrafish assay can provide a convenient approach to verify the disease risk of SMAD6 variants, and that zebrafish lacking smad6 function will be a tractable genetic model to study the role of Smad6 in development.

Using micro-computed tomography it is possible to detect the presence of pathologies which alter the lung's normal density. The density of the lungs can be altered depending on the amount of air, tissue, cells or fluid they contain. Using established mouse models of house dust mite (HDM) induced asthma, TGF-J31 induced pulmonary fibrosis (PF) and lipopolysaccharide (LPS) induced neutrophilic inflammation, this thesis examines if CT densitometry can distinguish between different pathophysiological processes. An airway segmentation method was applied to the CT images and data from these regions were assessed to determine: first, if pathologies can be detected compared to control animals; secondly, if pathological progression within each model can be measured; and finally, if it is possible to distinguish between the pathologies themselves. Lung histology and bronchoalveolar lavage fluid cytology, and total lung resistance (for the asthma model only) were assessed to confirm the disease models. The results showed that a healthy lung can be distinguished from a diseased lung in all three models. Pathological progression and resolution were also visible in the asthma and LPS groups. No changes were noted between the examined time points in the PF model. This corresponded to histological findings. It is also possible to distinguish between many of the pathologies based on the density profiles alone. Thus, CT densitometry affords a non-invasive method to longitudinally assess disease progression and resolution which is useful for the testing of novel therapeutics within the same subject. Regional CT density assessment, allows for the detection of localized pathologies around the airways which whole lung assessments may not be sensitive enough to detect. / Thesis / Master of Science (MSc)

The effective degree model has been applied to both SIR and SIS type diseases (those which confer permanent immunity and those which do not, respectively) with great success. The original model considers a large system of ODEs to keep track of the number of infected and susceptible neighbours of an individual. In this thesis, we use a generating function approach on the SIR effective degree model to transform the system of ODEs into a single PDE. This has the advantage of allowing the con- sideration of infinite networks. We derive existence and uniqueness of solutions to the PDE. Furthermore, we show that the linear stability of the PDE is governed by the same disease threshold derived by the ODE model, and we also show the nonlinear instability of the PDE agrees with the same disease threshold. / Graduate

Title: It’s easy to quit - I’ve done it hundreds of times: A literary study of the correlation between free will and substance abuse The purpose of this bachelor's thesis was to examine, through an integrative literary study, how selected previous research on the two theories, The Brain Disease Model of Addiction and The Choice Model of Addiction, describe the significance of free will in the development of and recovery from substance use disorder. The findings indicate that free will is significant in all stages of substance abuse. The study suggests that the discourse surrounding voluntariness and addiction ought to become more nuanced. The study notes that clients are easily influenced by treatment providers and thus conversations concerning disease versus free will should be approached cautiously by the treatment provider. The study suggests that social workers could benefit from critically examining their own stance regarding free will and addiction, including moral responsibility. Future research should continue developing screening methods to measure motivation levels in clients and to find the best treatment for each individual. / Syftet med denna kandidatuppsats var att genom en integrativ litteraturstudie undersöka hur utvald tidigare forskning om de två teorierna The Brain Disease Model of Addiction och The Choice Model of Addiction beskriver betydelsen av fri vilja när det gäller utvecklingen av och tillfrisknandet från ett substansberoende. Huvudresultatet visar att fri vilja är av betydelse i alla stadier av ett substansberoende. Studien tyder på att diskursen kring frivillighet och beroende torde blir mer nyanserad och att beroendeforskningen torde se beroende på ett spektrum. Studien konstaterar också att klienter är lättinfluerade av behandlare och därför bör samtal om sjukdom kontra fri vilja hanteras försiktigt av behandlaren i klientmöten. Socialarbetare som arbetar med beroende bör kritiskt granska sin egen ståndpunkt gällande beroendets frivillighet, inklusive moraliskt ansvar. Förslagsvis bör framtida forskning fortsätta att utveckla screeningmetoder för att bättre kartlägga motivationsnivån hos klienter och för att hitta den bästa behandlingsplanen för varje individ.

Malaria is one of the oldest parasitic diseases known to man, and the disease has played a role in shaping civilizations and the success of human populations over many centuries. While the malaria is well studied, it still remains a worldwide killer--claiming about 600,000 lives annually with children under the age of five representing a disproportionate population of those lethally infected. Malaria is caused by the protozoan parasite Plasmodium, which is introduced to the human body through the bite of a female Anopheles mosquito. The most lethal form of the disease is carried by the parasite Plasmodium falciparum, while the most widespread form of malaria is caused by Plasmodium vivax, the latter of which has a specific mode of entry and life cycle that makes it difficult to eradicate. The entry of P. vivax into human reticulocytes is based on the presence of the Duffy antigen chemokine receptor (DARC), which is uniquely absent in two-thirds of the Black population and populations of immediate African descent making it rare in the African region while endemic in Western and Asian countries. Inability to culture the parasite P. vivax in vitro and exhaustible tissue samples makes an accurate model of P. vivax malaria difficult to maintain ex vivo. The current study focuses on overcoming those limitations by modeling the mode of entry of P. vivax into patient-specific, induced pluripotent stem cell (iPSC)-derived erythrocyte-lineage cells by showing firstly that DARC is a measurable marker of susceptibility in vitro via FACS analysis, and that secondly, P. vivax cell culture limitations can be bypassed by creating a lentivirus designed to specifically infect DARC-expressing cells. To demonstrate the potency of this system, we show that a virus expressing the conserved region of the Duffy binding ligand, Duffy binding protein II (DBPII), can selectively infect peripheral blood mononuclear cells (PBMCs) that express DARC. Moreover, our current study focuses on the development of an iPSC-based disease model using patient samples derived from DARC expressing patients (DARC+) and DARC negative Sickle Cell Disease (SCD) patients (DARC-). We show that DARC+ iPSC-derived erythroid lineage cells express a transient population of DARC-expressing cells via FACS analysis, and we explore different protocols to stabilize this unique population. We hypothesize that DARC is a stage-specific marker for erythrocyte maturation, and we believe that any subset of cells expressing DARC consists of more mature erythrocyte-lineage cells. This study then, provides a novel platform by which to study malaria infection in a patient-specific manner while bypassing the limitations of culturing P. vivax in an in vitro culture system, as well as introducing a new way to measure erythrocyte maturation. Successful establishment of such a disease model has great implications for in-depth drug screenings for novel therapeutics that target the blood stage of the parasitic disease that were previously difficult to validate due to the limitations of currently existing models.

Salmon Pancreas Disease Virus (SPDV) is the only viral species of the genus Alphavirus, family Togaviridae, affecting fish. SPDV induces two conditions historically recognised independently as Pancreas disease (PD) and Sleeping disease (SD), affecting Atlantic salmon (Salmo salar L) and rainbow trout (Oncorhynchus mykiss), respectively. Infection by SPDV can lead to clinical disease with characteristic acinar pancreatic necrosis and a range of myopathies of the skeletal and heart muscle. Mortality is not a necessary outcome of the disease and usually is not significant. However, affected fish stop eating and therefore present a reduced growth rate and the disease can also leave visible lesions at the fillet level that lead to downgrading at slaughter. SPDV can affect in the fresh and sea water environments, but a higher and most relevant impact reported in the latter. Historically, PD has posed a significant challenge to the Atlantic salmon farming industry in the UK, as well as in other salmon producing countries. This thesis was developed and conducted at Marine Scotland Science (MSS), the Scottish National Reference Laboratory, with the aim to contribute to knowledge gaps identified by the industry and research communities. The focus was on development and improvement of in vivo and in vitro infection models to assist with host pathogen interaction studies. In vivo work was to establish an experimental challenge model to induce SPDV infection in a more natural way than by intra-peritoneal (IP) injection. The first step involved selection of an infective SPDV isolate through a comparative IP challenge study. An infective isolate was then used to establish a co-habitation challenge model in "post smolts", the sea-water stage predominantly affected by PD. Additionally, during this experiment assessment of viral tissue tropism along time and potential intra-subtype differences in infectivity was undertaken. In vitro work accounted for the more innovative part of this thesis with the development, optimization and application of an ex vivo cardiac primary culture originated from Atlantic salmon embryos. While fish origin aggregates of self-contracting cardiomyocytes had been previously isolated and suggested as a robust tool on human biomedical research and pharmacological and toxicology testing, paradoxically very little has been done to explore the approach of ex vivo primary cultures as a disease model with the specific goal for health issues affecting fish. The work involved an adaptation and refinement to produce salmon cardiac primary cultures (SCPCs). Once this was achieved, SCPCs could be kept under laboratory conditions with minimal maintenance for periods up to 6 months. Following this work, SCPCs were successfully challenged with different SPDV isolates as well as another cardiotropic viral agent (Infectious Salmon Anaemia, ISA). The kinetics of SPDV and ISA viral infection and one element of the immune response (i.e. expression of mx gene) were studied. As part of this study, the comparative response of SCPCs of diverse genetic backgrounds (i.e. IPN resistant vs. IPN sensitive) was also assessed. Differences were observed, which highlights potential usefulness of SCPCs to examine genotype-based differences in response to viral disease. Finally, SCPCs were used to examine the SPDV infection cycle ultrastructure by transmission electron microscopy (TEM). This work resulted in novel insights on the replication cycle of SPDV, drawing from the extensive literature in mammalian alphavirus work. With SPDV and other virus associated myocarditis severely affecting Atlantic salmon aquaculture at present, I believe that the SCPCs model represents the most relevant contribution of this PhD.

BACKGROUND: Calcific aortic valve disease (CAVD) is the most prevalent heart valve disease in the developed world, claiming almost 17,000 deaths annually in the United States. The lack of noninvasive therapeutics to slow or halt the disease warrants the need for further understanding of the pathobiological mechanisms of CAVD. A tri-laminar structure of aortic valve determines the biomechanical properties of its leaflets. Valvular endothelial cells (VECs) and interstitial cells (VICs) are responsible for valve structural integrity. Traditional two-dimensional culture conditions spontaneously activate the pathological differentiation of VICs making in vitro studies challenging. A monolayered three-dimensional (3D) hydrogel platform was recently developed as a novel in vitro culture system to study the phenotypic changes of VICs leading to microcalcification (early stages of calcification). This system, however, did not fully recapitulate the microenvironment of native valve tissues because of the lack of individual layer representations and endothelial coverage. Bioprinting technology, which allows precise and integrated positioning of cells, matrix, and biomolecules, may provide an innovative approach toward building a more biologically relevant 3D culture platform. OBJECTIVE: This study aims to lay the groundwork for building a multilayered 3D-bioprinted culture platform to study CAVD by first validating the use of bioprinting in monolayered cell-laden 3D hydrogel constructs. METHODS: Human VICs were isolated from patients undergoing valve replacement surgeries at Brigham and Women’s Hospital (Boston, MA) according to Institutional Review Board (IRB) protocols. VICs were expanded in culture medium containing growth factors for up to 6 passages and then encapsulated in hydrogels using 3D bioprinting technology. After encapsulation, VIC-laden 3D constructs were cultured in either normal or osteogenic conditions for 21 days. Microcalcification, cell proliferation, and cell apoptosis were evaluated using fluorescent staining and confocal microscopy. Results were compared with results from VIC-laden hydrogels made manually. RESULTS: An increase in microcalcification was observed throughout bioprinted VIC-laden hydrogel constructs cultured in osteogenic conditions for 21 days, whereas normal conditions developed negligible calcification signals. Cell proliferation and apoptosis were not significantly different between normal and osteogenic groups in bioprinted hydrogels. Cell-free hydrogels did not exhibit any microcalcification. Overall, bioprinted hydrogels showed less nonspecific background staining than handmade hydrogels, thus providing a better means for quantitative assessments of 3D culture platforms. CONCLUSION: Based on bioprinting technology, an improved monolayered cell-laden hydrogel platform was successfully established as a first step toward building an in vitro multilayered disease model for studying the pathobiological mechanisms of CAVD. The results in this study were consistent with current literature that proposes calcification as a cell-dependent, apoptotic-independent, and proliferation-independent pathway. / 2019-07-13T00:00:00Z

Orientador: Leticia Helena Theodoro / Coorientador: Valdir Gouveia Garcia / Banca: Leonardo Perez Faverani / Banca: Edilson Ervolino / Banca: Cassius Carvalho Torres Pereira / Banca: Mark Wainwright / Resumo: O presente estudo avaliou pela primeira vez "in vivo" os efeitos de três concentrações do butyl azul de toluidina (BuTB) como agente fotossensibilizador na terapia fotodinâmica antimicrobiana (aPDT), como terapia coadjuvante a raspagem e alisamento radicular (RAR), para o tratamento de periodontite experimental (PE) em ratos. A PE foi induzida por meio da instalação de um fio de algodão ao redor do primeiro molar inferior esquerdo. Posteriormente os animais foram aleatoriamente distribuídos em 7 grupos com 15 animais cada, através de uma tabela gerada por computador, de acordo com os seguintes tratamentos: RAR (n=15) - RAR seguido de irrigação local de solução salina fisiológica; BuTB-0,1 (n=15) - RAR seguido de aplicação local de BuTB na concentração de 0,1 mg/mL; aPDT-0,1 (n=15) - RAR seguido da aplicação local de BuTB na concentração de 0,1 mg/mL e irradiação com laser de diodo (LD) de InGaAlP (660 nm, 40 mW, 60 s, 2,4 J); BuTB-0,5 (n=15) - RAR seguido de aplicação local de BuTB na concentração de 0,5 mg/mL; aPDT-0,5 (n=15) - RAR seguido da aplicação local de BuTB na concentração de 0,5 mg/mL e irradiação com LD; BuTB-2,0 (n=15) - RAR seguido de aplicação local de BuTB na concentração de 2 mg/mL; aPDT-2,0 (n=15) - RAR seguido da aplicação local de BuTB na concentração de 2 mg/mL e irradiação com LD. Decorridos 7, 15 e 30 dias pós-tratamento, 5 animais de cada grupo foram submetidos à eutanásia. A área de furca dos molares foi submetida às análises histológica, histométrica... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The present study evaluated for the first time the effects of three concentrations of butyl toluidine blue (BuTB) as a photosensitizing agent on antimicrobial photodynamic therapy (aPDT), as adjuvant therapy to scaling and root planing (SRP), for the treatment of experimental periodontitis (EP) in rats. EP was induced by placing a cotton thread around the lower left first molar. Subsequently, the animals were randomly distributed into seven groups with 15 animals each, through a computer generated table, according to the following treatments: SRP (n = 15), SRP followed by local irrigation of physiological saline solution; BuTB-0.1 (n = 15), SRP followed by local application of 0.1 mg/mL BuTB; aPDT-0.1 (n = 15), SRP followed by local application of BuTB at 0.1 mg/mL concentration and irradiation with InGaAlP diode laser (DL) (660 nm, 40 mW, 60 s, 4 J); BuTB-0.5 (n = 15), SRP followed by local application of BuTB at 0.5 mg/mL concentration; aPDT-0.5 (n = 15), SRP followed by local application of BuTB at 0.5 mg/mL concentration and DL irradiation; BuTB-2.0 (n = 15), SRP followed by local application of BuTB at 2 mg/mL concentration; aPDT-2.0 (n = 15), SRP followed by local application of BuTB at 2 mg/mL concentration and DL irradiation. The animals (n=5) from each group were submitted to euthanasia at 7, 15 and 30 days post-treatment. The furcation area of the first lower molar was submitted to histological, histometric and immunohistochemical analyses to identify TGF-ß1, OCN an... (Complete abstract click electronic access below) / Doutor

This thesis examines the benefits and costs of screening for colorectal cancer in the context of an organised population screening programme. It uses microsimulation modelling to derive an optimally cost-effective screening protocol for various combinations of the available screening tests. ¶ First a mathematical model for the natural history of colorectal cancer is derived, based on analyses of Australian population and hospital-based cancer registries combined with data from published studies. Then a model for population based screening is derived based mainly on data from published screening studies, including the four major published randomised controlled trials of faecal occult blood test (FOBT) screening. These two models are used to simulate the application of a screening programme to the Australian population. The simulations are applied to a period of 40 years following 1990 (the study’s base year), with both costs and benefits discounted back to the base year at an annual rate of 3%.¶ The models are applied to simulating a population screening programme based on FOBT with a colonoscopy follow up of positive tests. This simulation suggests that the optimal application of such a programme would be to offer annual screening to people aged 50 to 84 years. Such a programme would lead to a cumulative fall in years of life lost to colorectal cancer (YLL) of 28.5% at a cost per year of life saved (YLS) of $8,987. These costs and benefits are consistent with those arising from other currently funded health interventions. They are also consistent with the cost per YLS which Australian governments appear willing to pay for health interventions when justified on the basis of cost-effectiveness. The fall in colorectal cancer deaths from this screening programme should be first detectable by a national monitoring system after around three years of screening. However the full benefits from screening would not be realised before around 30 years of screening.¶ These simulations are based on the standard guaiac FOBT, but the results suggest that significant cost-effective gains could be made by using the newer immunochemical FOBT. Further cost-effect gains could be made by offering sigmoidoscopy every five years in addition to annual FOBT.¶ The models are then applied to simulating population screening programmes using colonoscopy and sigmoidoscopy as primary screening tools. Offering colonoscopy every ten years to all people aged from 45 to 85 leads to an overall fall in cumulative YLL of 37.6%, at a cost of $15,585 per YLS. Offering sigmoidoscopy every three years to all people aged 40 to 85 leads to an overall fall in cumulative YLL of 29.1%, at a cost of $4,862 per YLS. Both of these cost and benefit results are also consistent with the cost per YLS which Australian governments appear willing to pay. The fall in deaths with colonoscopy screening would also be detectable after three years of screening but the fall with sigmoidoscopy screening would not be detectable until after six years of screening. Sigmoidoscopy would need around 35 years of screening to reach its potential gains while colonoscopy screening would not reach its full potential during the 40 year screening period.¶ Finally the models are applied to targeting people at higher risk of cancer. The results show that offering colonoscopy every five years to people at higher risk because of a family history of colorectal cancer is a cost-effective addition to the annual FOBT screening programme.¶ An earlier version of chapter two of this thesis has been published as Stevenson CE 1995. Statistical models for cancer screening. Statistical Methods in Medical Research; 4: 19–23.¶ An expanded version of chapter two, along with parts of chapter one, has been published as Stevenson CE 1998. Models of screening. In: Encyclopedia of Biostatistics. Armitage P, Colton T, eds. John Wiley and Sons Ltd, pp 3999–4022.